Fig. 1. Generation of the splicing-defective Tsix allele by gene targeting. (A) Scheme for generating the Tsix^SA allele. Genomic structure of the Xist locus and the targeting vector are shown. Tsix exons (gray boxes) are also aligned in parallel with Xist exons (black boxes). Differential polyadenylation of the spliced Tsix transcripts occurs by recognizing one of the polyA signals (pA) shown in the distal region of exon 4. Positions of the probes used for Southern blotting in B and D are also indicated. P, PstI; Pm, PmaCI; R, EcoRI; S, SacI; Xb, XbaI. The PmaCI site destroyed in the cloning process is indicated as (Pm). (B) Homologous recombination was confirmed by Southern blotting. SA177 is one of the three ES lines harboring the correct targeting event. Genomic DNA digested with PstI (left) and SacI (right) was probed with PE0.24 and probe D (Sado et al., 2001), respectively. (C) Comparison of the Tsix^SA allele and the Xist^1lox allele. The splicing acceptor site for exon 4 of Tsix, which is present in a 0.6-kb PmaCI-EcoRI fragment, is deleted in the Tsix^SA allele, but left intact in the Xist^1lox allele. (D) The presence of the respective mutation in the mouse was confirmed by Southern blotting. Tail DNA digested with PstI was probed with XXh0.7.